Immunostaining and in situ DNA analysis are useful tools in histological diagnosis and the study of tissue morphology. Immunostaining relies on the specific binding affinity of antibodies with epitopes in tissue samples, and the increasing availability of antibodies which bind specifically with unique epitopes present only in certain types of diseased cellular tissue. Immunostaining requiring a series of treatment steps conducted on a tissue section mounted on a glass slide to highlight by selective staining certain morphological indicators of disease states. Typical steps include pretreatment of the tissue section to reduce non-specific binding, antibody treatment and incubation, enzyme labeled secondary antibody treatment and incubation, substrate reaction with the enzyme to produce a fluorophore or chromophore highlighting areas of the tissue section having epitopes binding with the antibody, counterstaining, and the like. Each of these steps is separated by multiple rinse steps to remove unreacted residual reagent from the prior step. Incubations typically are conducted at around 40° C., while cell conditioning steps typically are conducted at somewhat higher temperatures, e.g. 90-100° C. In-situ DNA analysis relies upon the specific binding affinity of probes with unique nucleotide sequences in cell or tissue samples and similarly involves a series of process steps, with a variety of reagents and process temperature requirements.
Automated systems have been proposed to introduce cost savings, uniformity of slide preparation, and reduction of procedural human errors. Stross, W. et al, J. Clin. Pathol. 42: 106-112 (1989) describes a system comprising a series of baths positioned under the circumference of a circular, rotatable disc from which slide trays are suspended. The disc is lifted to lift slide trays from their baths, turned to position the slide trays above the next consecutive bath, and lowered to immerse the slide trays in the baths. This operation can be automated with suitable timers and switches. This system exposes each of the slides to the same treatment and relies on dipping for application of reactants and rinsing.
Stark, E. et al, J. Immunol. Methods. 107: 89-92 (1988) describes a microprocessor controlled system including a revolving table or carousel supporting radially positioned slides. A stepper motor rotates the table, placing each slide under one of the stationary syringes positioned above the slides. A predetermined volume of liquid, determined by a dial, is delivered to a slide from each syringe. Microprocessor controls are provided.
Cosgrove, R. et al, ACL. pp 23-27 (December, 1989) describe an immunostaining apparatus for auto-pipetting reagents into a slide well from a carousel holding up to 18 reagent vials. Below each well, a coverplate spaced from the surface of each slide provides cover and defines a reagent flow channel. The slides are suspended at a steep angle. Reagent from the well flows downward over the slide surface. A row of slides are suspended for sequential treatment. Washing is accomplished by a 3 to 4 minute continuous running wash over the sample, yielding an estimated 20:1 wash/reagent ratio.
Brigati, D. et al, J. Histotechnology 11: 165-183 (1988) and Unger, E. Brigati, D. et al, et al, J. Histotechnology. 11: 253-258 (1988) describe the Fisher automated work station using capillary gap technology. A coverplate is placed over the slide, forming a capillary gap. Liquid is introduced into the capillary gap by placing the lower edge of the plate-slide pair in a liquid. Liquid is removed by placing the lower edge of the plate-slide pair on a blotter. The system is further described in U.S. Pat. Nos. 4,777,020, 4,798,706 and 4,801,431. The previously known devices are listed in their performance and unable to satisfy the needs for automated, high precision immunohistology.
The foregoing discussion of the prior art derives in large part from U.S. Pat. No. 5,654,200 to Copeland et al., who describe an automated biological processing system comprising a reagent carousel cooperating with a sample support carousel to apply a sequence of preselected reagents to each of the samples with interposed mixing, incubating, and rinsing steps cooperating therewith. This patented automated biological processing system, which is available from Ventana Medical Systems, Inc. of Tucson, Ariz. includes a slide support carousel having a plurality of slide supports thereon and drive means engaging the slide support carousel for consecutively positioning each of a plurality of slide supports in a reagent receiving zone. The reagent carousel has a plurality of reagent container supports thereon and drive means engaging the reagent carousel for rotating this carousel and positioning a preselected reagent container support and associated reagent container in a regent supply zone. The apparatus has a reagent delivery actuator means positioned for engaging a reagent container positioned on a container support in the reagent supply zone and initiating reagent delivery from the reagent container to a slide supported on a slide support in the reagent receiving zone.
FIG. 1, which largely corresponds to FIG. 3 of U.S. Pat. No. 5,654,200 is a partial exploded isometric view of an automated biological processing system, with the cabinet, liquid and air supply tubing and electrical wiring omitted in the drawings for the purposes of clarity.
The apparatus has an upper section 2, intermediate section 4 and lower section 6. In the upper section 2, reagent bottle support carousel 10 is mounted for rotation about its central axis on upper support plate 8. Reagent bottles 12 required for the immuno-histochemical reactions to be conducted during slide treatment cycle are supported by the carousel 10, mounted in reagent bottle receptors 11. These receptors 11 are configured to receive volumetric pump outlet tubes (not shown). The receptors 11 are preferably equally spaced in a circular pattern axially concentric with the carousel axis. The number of receptors 11 provided should be sufficient to accommodate the number of different reagent bottles 12 required for a cycle or series of cycles. The carousel 10 is rotated by the stepper motor 14 and drive belt 16 to a position placing a selected reagent bottle 12 in the reagent delivery position under an air cylinder reagent delivery actuator 18 over a slide to be treated with reagent. Reagent tray motor driver 20 is connected to stepper motor 14.
The intermediate section 4 comprises support plate 22 upon which the slide support carousel 24 is rotatably mounted. The carousel 24 supports slide supports 26. In the intermediate section 4, a stepper motor 48 rotates the slide support carousel 24, engaging drive belt 25 engaging the perimeter of the slide support carousel 24. Splash guard 50 is a wall which surrounds the sides, back and part of the front of the carousel 24, and contains liquid spray and droplets produced in the processing. Splash guard 50 extends upward from the intermediate plate 22 to a position adjacent the upper plate 8, leaving an air flow gap between the upper edge of the splash guard 50 and the underside of the plate 8. Lower section 6 includes slide carousel stepper motor driver 72 and relay 74, power supplies 76 and 78, and control systems all mounted on plate 40.
Referring to FIGS. 2 and 3, slide support 26 comprises a molded plastic base 80 on which is mounted a metal plate 82. An electrical resistance heater shown in phantom at 84 is mounted in direct contact to the underside of metal plate 82. Corner pins 86 locate a specimen carrying glass slide 88 on the surface of metal plate 82. Metal plate 82 has a top surface that is essentially flat and smooth. Flatness and smoothness facilitates glass plate position stability and thermal conduction uniformity.
In practice, water and other fluids employed in the slide processing may spill over the edges of the slides, and work their way under the slides where the fluids may boil, causing the slides to “pop” or dislocate. Moreover, since heater surfaces are not perfectly flat, in order to insure good thermal contact between metal plate 82 and glass slide 88, a thin layer 90 of oil may be applied to the top surface of metal plate 82. However, using oil as an interfacial heating medium, may exacerbate the problem of slide popping or dislocation due to gas formation from water or other fluid getting under the slide, mixing with the oil and then boiling off in an uncontrolled fashion. Dislocation of a slide may cause that slide to set up on a post, thereby compromising the processing of that one slide, or in a worse case scenario result in a domino or train wreck effect where the one dislocated slide hits a neighboring slide causing that slide to dislocate, and so forth.